Oligomers formed by the amyloid beta peptide, A-beta, have been shown to be neurotoxic, potently permeabilize biological membranes and are believed to be critical players in Alzheimers disease (AD). Applying traditional approaches to study these oligomers and the origin of their toxicity is challenging because ensemble averaging masks low amounts of transient intermediates and hinders the resolution of species heterogeneity especially when bound to membranes. In this two year ARRA supported project we propose to apply novel approaches based on single molecule microscopy (SMM), that are uniquely suited for these studies, to focus on the interactions between A-beta and neuronally-derived membranes to address the following specific aims: Aim 1: To determine the time evolution of A-beta oligomers in solution and identify the oligomers that bind to neuronal membranes and permeabilize them. We will characterize the interactions between solution-generated A-beta oligomeric species and membrane preparations made from brain extracted lipids as well as membrane patches excised from neuronal cells. These measurements will test the hypothesis that at elevated peptide concentrations, toxic oligomers form directly in solution and bind to and permeabilize the membrane without the need for additional growth. Aim 2: To determine the mechanism of Abeta oligomer assembly on the surface of membranes from neuronal cells, characterize the size of the oligomers at the onset of permeabilization and follow their subsequent evolution. Work on this Aim will allow us to: a) determine the effect of the native membrane environment on the oligomer assembly process and on the permeabilization power of these oligomers (relative to the solution generated oligomers in aim 1);b) determine how the membrane facilitates the formation of toxic oligomers at the low physiological concentrations of A-beta. One of the major missions of the ARRA funding is the creation of new jobs and the retention of current ones. In this request the additional support will be used to speed up the tempo of research, and will allow us to create three new positions (two GSRA and one postdoctoral) and to retain two experienced researchers who would otherwise be laid off.